Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement

被引：92

作者：

Garrett, JB ^{[1
]}

Kretz, KA ^{[1
]}

O'Donoghue, E ^{[1
]}

Kerovuo, J ^{[1
]}

Kim, W ^{[1
]}

Barton, NR ^{[1
]}

Hazlewood, GP ^{[1
]}

Short, JM ^{[1
]}

Robertson, DE ^{[1
]}

Gray, KA ^{[1
]}

机构：

[1] Diversa Corp, San Diego, CA 92121 USA

来源：

APPLIED AND ENVIRONMENTAL MICROBIOLOGY | 2004年 / 70卷 / 05期

关键词：

D O I：

10.1128/AEM.70.5.3041-3046.2004

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this: study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62degreesC for 1 h and 27% of its initial activity after 10 min at 85degreesC, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.

引用

页码：3041 / 3046

页数：6

共 30 条

[1] HIGH-RESISTANCE OF ESCHERICHIA-COLI RIBONUCLEASE HI VARIANT WITH QUINTUPLE THERMOSTABILIZING MUTATIONS TO THERMAL-DENATURATION, ACID DENATURATION, AND PROTEOLYTIC DEGRADATION [J].